The Ldb1 complex, including GATA-1 and TAL1, mediates long range interaction between the beta-globin locus control region (LCR) and gene in adult mouse erythroid cells. In a human erythroid cell model in which fetal gamma-globin genes can be robustly re-activated by cytokines, we showed that the Ldb1 complex mediates gamma-globin/LCR proximity through a site 3 to the genes. Reactivation of these genes is therapeutic in beta-thalassemia and sickle cell disease and understanding the underlying mechanisms is critical. The results suggested that when Eto2 associates with the Ldb1 complex gamma-globin/LCR interactions and expression are reduced. To identify factors functioning downstream of Eto2 that may regulate switching from gamma to beta-globin transcription, we performed RNA-Seq in control and Eto2 depleted human erythroid cells. We identified and are studying over 400 genes (Padj < 0.05) that were mis-regulated in the absence of Eto2. To determine if Eto2 is required for hemoglobin switching, we established a model of this process in CD34+ umbilical cord blood cells. Knock down of ETO2 during differentiation of these cells supports the idea that Eto2 is a repressor of gamma-globin expression and a regulator of human hemoglobin switching. In addition, RNA-Seq experiments identified nearly 4,000 genes (Padj < 0.01) that were differentially regulated during switching. Of these, over 200 are known erythroid fingerprint genes and are under study. The Ldb1 site 3 to the gamma-globin genes that is involved in LCR looping is within sequences of BGL3, a non-coding RNA transcript. BGL3 transcription parallels that of the gamma-globin genes in various erythroid cell models although at a much lower level. We are studying the function of BGL3 during hemoglobin switching. We are following BGL3 and gamma-globin transcripts by RT-PCR and RNA-FISH. To investigate whether the expression of gamma-globin depends on the BGL3 transcript or transcription per se, we are knocking down or over-expressing BGL3 and examining the effect on gamma-globin long range LCR interactions and transcription. We are also targeting deletion of BGL3 sequences and investigating its protein interactome. We are using homologous recombination in mouse ES cells study how individual globin gene establish stage specific enhancer communication. We targeted a region upstream of the mouse embryonic epsilon y gene and a second region upstream of the adult beta-major gene on one allele in ES cells by homologous recombination. Recombinase mediated cassette exchange was used to insert chromatin insulator human HS5 or a transcription terminator in these two positions. In differentiating ES cells, insertion of the transcription terminator or hHS5 between the LCR and downstream genes inhibits embryonic ey gene activation in an allele-specific fashion but neither insertion affects adult beta-globin gene expression. We have undertaken blastocyst injection of our successfully targeted ES cells. The mouse lines we are producing will be used to understand how long range interactions and gene transcription in the beta-globin locus are perturbed by the insertions during erythroid development. These experiments are novel since they alter chromatin organization in an endogenous locus.